Animals ; Apoptosis ; Cell Differentiation ; Cell Proliferation ; Fluoride Poisoning ; metabolism ; pathology ; Fluorosis, Dental ; metabolism ; pathology ; Hedgehog Proteins ; genetics ; metabolism ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteoclasts ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Signal Transduction ; Stomach Neoplasms ; metabolism ; pathology

Animals ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Differentiation ; Humans ; Mouth Neoplasms ; metabolism ; pathology ; Oral Medicine ; Osteoblasts ; cytology ; Osteoclasts ; cytology ; Tooth ; metabolism ; Wnt Proteins ; metabolism ; physiology ; Wnt Signaling Pathway ; physiology ; beta Catenin ; metabolism

This study was aimed to investigate the biological characteristics of osteoblasts from patients with myelodysplastic syndrome (MDS) and their supportive capacity for hematopoiesis in vitro. A two-dimensional culture system was constructed by using osteoblasts derived from human marrow mesenchymal stem cells (MSC); MSCs were isolated from bone marrow of MDS patients and normal individuals and were cultured; the third passage of MSCs were induced into osteoblasts which were treated with mitomycin C and confluenced into a feeder layer. Ficolled bone marrow mononuclear cells were obtained from normal individuals and seeded into the two-dimensional culture system to culture in vitro without exogenous cytokines. By using colony-forming assay, the ability of the two-dimensional system to culture HPCs was observed. The cytokine expression of osteoblasts from MDS patient bone marrows in mRNA level was detected by RT-PCR and was compared with human osteoblast cell line hFOB1.19. The results showed that the osteoblasts from MDS patients could support short-term survival of GM-CFC in condition without exogenous cytokines, that is, osteoblasts played a crucial role in regulation of HPC growth. The results of RT-PCR clearly demonstrated that the osteoblast cell line hFOB1.19 expressed SCF, IL-6, SDF-1alpha, G-CSF and GM-CSF. The same expression patterns of above cytokines were also seen in osteoblasts derived from BM-MSCs of MDS patients and normal individuals, but these cells did not express GM-CSF. It is concluded that the biological characteristics of osteoblasts from bone marrow of MDS patients are generally not different from those of osteoblasts from normal bone marrow. Both of them can support GM -CFC to form colonies in vitro, it may be associated with expressing important related cytokines by osteoblasts.
Cytokines ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-6 ; metabolism ; Myelodysplastic Syndromes ; metabolism ; pathology ; Osteoblasts ; metabolism ; physiology ; RNA, Messenger ; metabolism ; Stem Cell Factor ; metabolism

This paper is aimed to explore the effects of mechanical stimulation on proliferation and differentiation of osteoblasts. Cultured MG-63 osteoblast-like cells were strained by the four-point bending cell mechanics loader. In the study, we observed the effects of different magnitudes and duration of mechanical strain on the markers of proliferation and differentiation in osteoblasts. The protein levels and Alkaline phosphatase (ALP) activity were determined by western blot and alpha-nitrophenyl phosphate assay respectively. The mineralization nodules were stained using Alizarin Red-S method. We found: (1) the expression of proliferating cell nuclear antigen (PCNA), ALP activity in strained group were significantly increased compared to those in the control group, but the role did not increase with the increase of the magnitude of the stimulation; and (2) under appropriate stimulation (2000 microstrain), the expression of PCNA, COL I protein and ALP activity increased gradually with the increase of loading time, and appropriate stimulation promoted the formation of mineralization nodules. It indicated that appropriate mechanical stimulation could promote proliferation and differentiation of osteoblasts.
Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Osteoblastoma ; pathology ; Osteoblasts ; cytology ; Proliferating Cell Nuclear Antigen ; metabolism ; Stress, Mechanical

OBJECTIVETo observe the effect of Gusongbao (GSB) on proliferation and metabolism of osteoblast cultured in vitro.

METHODSOld rats, aged 18 months, were given GSB 1.5 g/kg, twice a day for 3 days by intragastric perfusion. Blood of the rats was collected 1 hr after the final perfusion to isolate serum for preparing, with D8900 medium, the culture media containing 7.5% or 15% GSB, which was used to culture osteoblast for 24 hrs. Besides, D8900 media containing 7.5% or 15% old rats'serum without medication, containing 20 mumol/L, sodium fluoride, and simple D8900 medium were prepared for control. The cell proliferation was detected by MTT method, and the changes of Ca2+ concentration and ALP content in supernatant of culture were also observed.

RESULTSThe osteoblast proliferation cultured in GSB serum containing medium was significantly increased than those cultured in the other control media (P < 0.01), at the same time, the Ca2+ consumption increased and the ALP content elevated significantly (P < 0.05, P < 0.01).

CONCLUSIONGSB could promote the DNA synthesis, increase the utilization of Ca2+ and accelerate the growth and proliferation of osteoblast.

Animals ; Calcium ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; Female ; Male ; Osteoblasts ; cytology ; metabolism ; Osteoporosis ; pathology ; Rabbits ; Rats ; Rats, Wistar

OBJECTIVETo culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM).

METHODSBone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, β-sodium glycerophosphate and vitamin C, which were identified by alkaline phosphatase staining, Von Kossa's staining. The CCL3 receptor expression was evaluated by flow cytometry. The morphology and quantity of osteoblast were observed after exposure to CCL3.

RESULTSBone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa's. MM-derived osteoblasts expressed higher levels of CCR1 (74.48 ± 7.31)%, compared with normal controls (48.35 ± 8.81)%. Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls.

CONCLUSIONBone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.

Adult ; Aged ; Cells, Cultured ; Chemokine CCL3 ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; pathology ; Osteoblasts ; cytology ; drug effects ; metabolism ; Receptors, CCR1 ; metabolism ; Young Adult

The study was purposed to explore the role of mesenchymal stem cells (MSCs) in the pathogenesis of bone disease particularly observed in multiple myeloma (MM), the biological features of marrow derived MSCs from patients with MM have been investigated. Marrow aspirates were harvested from 11 newly diagnosed patients with MM and 5 normal adults and MSCs were isolated and culture-expanded by the cell properties of adherence to plastic flasks, The phenotype was analyzed by flow cytometric technique. The proliferation of MSCs was observed by MTT assay and their differentiation capacities into osteoblasts and adipoblasts were assessed with lineage-specific histochemical staining. The concentrations of IL-6 and SCF in the culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MSC culture supernatants were collected and MTT assay was performed to evaluate their support on the proliferation of an MM cell line SKO007 cells. The results showed that bone marrow-derived MSCs from MM patients were homogeneously positive for CD29, CD73, CD166 and HLA-ABC and negative for hematopoietic cell marker CD45 and endothelial cell marker CD31, the phenotype of which was similar to that of marrow counterparts from normal adults. MTT assay indicated that MSCs from MM patients or normal adults proliferated at similar rates. MSCs from MM patients occupied in vitro osteogenic and adipogenic capacity as those from normal adults. The levels of IL-6 and SCF in culture supernatant were greatly up-regulated in MM patients by ELISA assay. Furthermore, MSC culture supernatants from MM bone marrow displayed enhanced activity to promote the proliferation of SKO007 cells. It is concluded that marrow-derived MSCs from bone marrow of MM patients are normal in their proliferation and differentiation capacities, and myeloma bone disease may not be ascribed to the differentiation of MSCs while the elevated secretion of IL-6 and SCF may provide necessary cues for the survival of malignant myeloma cells.
Bone Marrow Cells ; pathology ; Cell Differentiation ; Cells, Cultured ; Female ; Humans ; Interleukin-6 ; analysis ; Male ; Mesenchymal Stromal Cells ; metabolism ; pathology ; Middle Aged ; Multiple Myeloma ; pathology ; Osteoblasts ; cytology ; Stem Cell Factor ; analysis

OBJECTIVETo investigate the role of transcription factor special AT-rich binding protein 2 (SATB2) in the osteoblasts differentiation of bone marrow stromal cells (BMSC) in vitro.

METHODSRats bone marrow stromal cells were isolated by Percoll sedimentation and the cells were placed and allowed to attach for three times. After passages, expression plasmid pBABE-hygro-satb2 was constructed, then transfected into BMSC. BMSCs were inoculated in conditioned medium and osteogenic factors were detected by western blotting and reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe morphological observation of BMSC showed either spindle or polygonal pattern. The cellular phenotypic marker of the third passage was CD29 positive and CD34 negative. The growth curve possessed "S" pattern. The intensity of calfilication in BMSC was higher in SATB2 transfection group (IA value 125974 ± 241) than that in the control groups (IA value 178486 ± 406). Moreover, cell migration rate increased in SATB2 transfection group [width of scratch (0.72 ± 0.01) mm] compared with control group [width of scratch (0.83 ± 0.03) mm]. In addition, the mRNA expression of osteogenic factors runt-related transcription factor 2, Osterix, activating transcription factor 4, integrin-binding sialoprotein were upregulated.

CONCLUSIONSCells cultured with this method have general biological characteristics and osteogenic differentiation potential in vitro. SATB2 can promote osteoblasts differentiation of BMSC.

Activating Transcription Factor 4 ; metabolism ; Animals ; Bone Marrow Cells ; metabolism ; pathology ; Cell Differentiation ; Cell Movement ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Integrin-Binding Sialoprotein ; metabolism ; Male ; Matrix Attachment Region Binding Proteins ; genetics ; metabolism ; Osteoblasts ; cytology ; Osteogenesis ; Plasmids ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; metabolism ; pathology ; Thy-1 Antigens ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

MicroRNAs (miRNAs) have recently been recognized to have a role in human orthopedic disorders. The objective of our study was to explore the expression profile and biological function of miRNA-17-5p (miR-17-5p), which is well known to be related to cancer cell proliferation and invasion, in osteoblastic differentiation and in cell proliferation. The expression levels of miR-17-5p in the femoral head mesenchymal stem cells of 20 patients with non-traumatic osteonecrosis (ON) and 10 patients with osteoarthritis (OA) were examined by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, the interaction between miR-17-5p and SMAD7 was observed. We found that in non-traumatic ON samples the level of mature miR-17-5p was significantly lower than that of OA samples (P=0.0002). By targeting SMAD7, miR-17-5p promoted nuclear translocation of beta-catenin, enhanced expression of COL1A1 and finally facilitated the proliferation and differentiation of HMSC-bm cells. We also demonstrated that restoring expression of SMAD7 in HMSC-bm cells partially reversed the function of miR-17-5p. Together, our data suggested a theory that dysfunction of a network containing miR-17-5p, SMAD7 and beta-catenin could contribute to ON pathogenesis. The present study prompts the potential clinical value of miR-17-5p in non-traumatic ON.
Adult ; Base Sequence ; Bone Morphogenetic Protein 2/metabolism ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Female ; *Gene Expression Regulation ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; Osteoarthritis/genetics/metabolism/pathology ; Osteoblasts/*cytology/metabolism/*pathology ; Osteogenesis ; Osteonecrosis/*genetics/metabolism/pathology ; Signal Transduction ; Smad7 Protein/*genetics/metabolism ; beta Catenin/metabolism

The cajanine (longistylin A-2-carboxylic acid) is isolated and identified from extracts of Cajanus cajan L. (ECC) , which structure is similar to diethylstilbestrol. The regulation properties of the cajanine and other four extracts of Cajanus cajan L. (32-1, 35-1, 35-2, and 35-3) were tested in human osteoblast-like (HOS) TE85 cells and marrow-derived osteoclast-like cells. By using MTT assay to test the change of cell proliferation, 3H-proline incorporation to investigate the formation of collagen, and by measuring alkaline phosphatase (ALP) activity, bone formation in HOS TE85 cell was evaluated after pretreated for 48 hours. Bone marrow cells were cultured to examine the derivation of osteoclast cells (OLCs), which were stained with tartrate-resistant acid phosphatase (TRAP). The long term effect (pretreated for 18 days) on promoting mineralized bone-like tissue formation was tested by Alizarin red S staining in HOS TE85 cells. After the treatment with cajanine (1 x 10(-8) g x mL(-1)) for 48 hours, cell number increased significantly (57.7%). 3H-Proline incorporation also statistically increased (98.5%) in those cells. Significant change of ALP activity was also found (P < 0.01) in 35-1 and 35-3 treated cells (they were 66.2% and 82.4% in the concentration of 1 x 10(-8) g x mL(-1), respectively). The long term (18 days) effects of 32-1 and 35-3 on promoting mineralized bone-like tissue formation in HOS TE85 cell were obvious. There were much more red blots over the field of vision compared with that of control group. After the treatment of cajanine, derived-osteoclast cells appeared later and much less compared with control. The inhibition of cajanine was 22.8% while it was 37.9% in 32-1 treated cells in the dose of 1 x 10(-7) g x mL(-1). It is obvious that cajanine and ECCs promoted the osteoblast cells proliferation and mineralized bone-like tissue formation in HOS TE85 cells, while inhibited derivation of osteoclast cells. All of these suggested that cajanine has the estrogen-like action on osteoblast and osteoclast, which could be developed as anti-osteoporosis drugs.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Bone Neoplasms ; metabolism ; pathology ; Cajanus ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Diethylstilbestrol ; analogs & derivatives ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Osteoblasts ; drug effects ; Osteoclasts ; cytology ; metabolism ; Osteogenesis ; drug effects ; Osteosarcoma ; enzymology ; pathology ; Phytoestrogens ; isolation & purification ; pharmacology ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar